This proposal is to investigate the development of the hemopoietic and lymphoid systems during early embryogenesis in the clawed toad, Xenopus laevis. Amphibians will be used in these experiments because they are accessable at all developmental stages, induction of triploidy provides a permanent cytogenetic marker, presumptive hemopietic rudiments can be grafted prior to circulation, hemopoietic stem cells can be isolated and cultured prior to differentiation and isogenic strains provide a means of investigating the basis of cell-cell interactions.
Specific aims related to the hemopoietic system are to examine the basis for regulatory interaction occurring between dorsal and ventral stem cell compartments and the extent to which differentiation programs can be changed by the microenvironment.
Aims related to the lymphoid system are to examine the divergence of lymphoid stem cells from hemopoietic stem cells, to determine the frequency of lymphoid progenitors in the dorsal and ventral stem cell compartments, and to assess whether a bipotential thymocyte-accessory cell progenitor is involved in development of the self-non-self repertoire.
The third aim i s to examine the cell surface of differentiating stem cells and to determine if there are any changes in the differentiation antigens during progression from uncommitted stem cell to lymphoid progenitors. The methods which will be used include serum free culture of isolated stem cell compartments explanted from 17-20 hour old embryos, reciprocal transplantation of cytogenetically labeled stem cell compartments, and limiting dilution translantation of cytogenetically labeled stem cells. Skin grafting and specific cytotoxicity will be used to examine tolerance and alloreactivity during development, FACS analysis will be used to determine the frequency of cytogenetically marked cells, and monoclonal antibodies will be developed in order to examine differentiation antigens on the cell surface during development.
